Interferon analogs

ABSTRACT

The invention relates to the field of medicine. Among others, it relates to biologically active analogs of interferons (IFNs) which show less unwanted side-effects and to the therapeutic uses thereof. Provided is an IFN analog, wherein the moiety mediating binding to its natural receptor is at least functionally disrupted and wherein the analog comprises a signaling moiety capable of mediating intracellular IFN activity, said signaling moiety being provided at its N-terminus, optionally via a linker, with at least one targeting domain capable of binding to a cell surface receptor other than the IFN receptor.

The invention relates to the field of medicine. Among others, it relatesto biologically active analogs of interferons (IFNs) which show lessunwanted side-effects and to the therapeutic uses thereof.

About ten distinct IFNs have been identified in mammals; seven of thesehave been described for humans. They are typically divided among threeIFN classes: Type I IFN, Type II IFN, and Type III IFN. All type I IFNsbind to a specific cell surface receptor complex known as the IFN-αreceptor (IFNAR) that consists of IFNAR1 and IFNAR2 chains. The type Iinterferons present in humans are IFN-α, IFN-β and IFN-ω). Interferontype II binds to IFNGR. In humans this is IFN-γ. By interacting withtheir specific cell surface receptors, IFNs activate signal transducerand activator of transcription (STAT) complexes; STATs are a family oftranscription factors that regulate the expression of certain immunesystem genes. Some STATs are activated by both type I and type II IFNs.However each IFN type can also activate unique STATs (Platanias, L. C.2005 Nature reviews. Immunology 5 (5): 375-386).

IFNs belonging to all IFN classes are very important for fighting viralinfections. Although they are named after their ability to “interfere”with viral replication within host cells, IFNs have other functions:they activate immune cells, such as natural killer cells andmacrophages; they increase recognition of infection or tumor cells byup-regulating antigen presentation to T lymphocytes; and they increasethe ability of uninfected host cells to resist new infection by virus.Certain host symptoms, such as aching muscles and fever, are related tothe production of IFNs during infection.

IFNγ is a pleiotropic cytokine produced by the activated immune cells.It acts through the IFNγ receptor that is expressed nearly on all celltypes, however it displays a strict species specificity. IFNγ has beenapplied for the treatment of immunological, viral and cancer diseases(Younes and Amsden, J Pharm Sci 2002) with significant effects. Inaddition, several studies have demonstrated the potential role of IFNγin renal and liver fibrosis (Kidney Int. 1999; 56:2116-27, Hepatology1996 23:1189-99).

Unfortunately, the short circulation half-life and undesirable systemicside effects of currently available interferons have limited itsclinical application and even halted clinical trials. Many attempts havebeen made to circumvent these problems e.g. by incorporating IFN_(Y)into liposomes, microspheres and elastomers (Pharm Res. 2000 17:42-8,Pharm Res. 1996 13:1464-75, J Control Release. 2005 102:607-17).Cell-specific delivery approaches have not been used so far. This is notsurprising in view of the fact that such an approach is generally deemedimpossible for cytokines which need to be delivered to their ownreceptors to elicit a biological effect, so that they always end up intarget cells that express these receptors. Delivery to other targetreceptors will therefore in most cases be useless and at best lead touptake in other cell types causing loss of activity or furtherdiversification of adverse effects.

However, the present inventors recognized that the structure of INFoffers unique targeting opportunities since the molecule contains areceptor binding moiety which is species-specific and a signaling moietywhich is non-species specific and which acts intracellularly. It wassurprisingly found that that the unique structure of INF_(Y) allowsdelivery of the signaling moiety of IFN_(Y) to another target receptor,provided that this new target receptor allows intracellular release ofthis signaling moiety and subsequent activation of theintracellular/nuclear INF_(Y) signaling pathway. For example, atruncated IFNγ mimetic targeted to the Platelet Derived Growth Factor(PDGF) receptor displayed significantly less systemic side effects in anacute liver injury mouse model when compared to full length IFNγ ornon-targeted IFNγ mimetic.

Accordingly, the invention relates to an analog of interferon (IFN),wherein the moiety mediating binding to its natural receptor is at leastfunctionally disrupted and wherein the analog comprises a signalingmoiety capable of mediating intracellular IFN activity, said signalingmoiety being provided at its N-terminus, optionally via a linker, withat least one targeting domain capable of binding to a cell surfacereceptor other than the IFN receptor. Preferably, the analog is an IFNgamma (IFNγ) analog.

Interferon analogs provided with a heterologous targeting moiety areknown in the art. WO2008/068621 discloses a conjugation product of IFNγand a targeting moiety, such as a tumor targeting moiety or a tumorvasculature targeting moiety. The prior art only discloses conjugationto full length IFNγ, which will still bind to its native receptorcausing adverse effects.

EP0844252 aims to provide cyclic peptides and their preparation process,which allow subsequent chemical grafting on coupling on said cyclicpeptides, i.e. their attachment to a solid support, to a high molecularweight compound, to a marker and/or to other cyclic peptides, in orderto provide new or improved biotechnological applications, particularlyin the field of affinity chromatography, immunisation, development ofdiagnostic tests, vaccines and pharmaceutical compositions. It teachesthe coupling of cyclic peptide ligands to biological molecules, amongothers to interferons. No mention is made in EP0844252 about the use ofa truncated IFN molecule lacking a functional binding domain to itsnatural receptor.

An interferon analog of the present invention has clear and unexpectedadvantages over the use of a (modified) full length interferon, not onlywith respect to its therapeutic use (less side-effects, increasedefficacy, reduced antigenicity because of lower molecular weight) butalso to its production process (recombinant synthesis).

The functional disruption of the moiety mediating binding to its naturalreceptor can be achieved by deleting, inserting, substituting one ormore relevant amino acid residues in the receptor binding domain.Interferon receptor binding domains have been identified and are knownin the art. For example, binding of murine IFNγ to the INFγ receptor canbe abolished by at least partially deleting the first 40, 30 or 20N-terminal residues.

In one embodiment, the analog is a truncated IFNγ polypeptide which onlycontains the residues involved in intracellular signaling. As usedherein, intracellular signaling may comprise nuclear translocationand/or anti-viral activity.

Preferably, the signaling moiety mediating intracellular activitycomprises a polybasic nuclear localization signal (NLS) motif as foundin the C-terminus of human and murine IFN (Subramaniam et al. 2000, J.Cell Science 113, 2771-2781). This NLS motif is thought to form acomplex with a signal transducer and activator of transcription (STAT);STATs are a family of transcription factors that regulate the expressionof certain immune system genes. Some STATs are activated by both type Iand type II IFNs. However each IFN type can also activate unique STATs(Platanias, L. C. 2005 Nature reviews. Immunology 5 (5): 375-386) STATactivation initiates the most well defined cell signaling pathway forall IFNs, the classical Janus kinase-STAT (JAK-STAT) signaling pathway.In this pathway, JAKs associate with IFN receptors and, followingreceptor engagement with IFN, phosphorylate both STAT1 and STAT2. As aresult, an IFN-stimulated gene factor 3 (ISGF3) complex forms—thiscontains STAT1, STAT2 and a third transcription factor called IRF9—andmoves into the cell nucleus. Inside the nucleus, the ISGF3 complex bindsto specific nucleotide sequences called IFN-stimulated response elements(ISREs) in the promoters of certain genes; this induces transcription ofthose genes.

An analog provided herein may comprise the polybasic NLS motif comprisesthe amino acid sequence (R)KRXRS(R), (SEQ ID NO: 1) wherein X is anyamino acid residue, preferably wherein X is R, K, S or T. Preferably,the NLS motif is present at the C-terminal end of the analog. In oneembodiment, it comprises the sequence, preferably the C-terminalsequence RKRKRSR (SEQ ID NO: 2), KSKRSR (SEQ ID NO: 3), KRTRS (SEQ IDNO: 4) or KRTRSQ (SEQ ID NO: 5).

In a specific aspect, the signaling moiety comprises or consists of asequence selected from the group consisting of

(a) the amino acid sequence KFEVNNPQVQRQAFNELIRVVHQLLPESSLRKRKRSR (SEQID NO: 6); (b) the amino acid sequenceYSVTDLNVQRKAIHELIQVMAELSPAAKTGKRTRSQ (SEQ ID NO: 7) orYSVTDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQ (SEQ ID NO: 8);

(c) the amino acid sequence AKFEVNNPQIQHKAVNELIRVIHQLSPESSLRKRKRSRC (SEQID NO: 9)

(d) a stretch of at least 10, preferably at least 15, contiguous aminoacids of the sequence of (a), (b) or (c);

(e) an amino acid sequence showing at least 70%, preferably at least80%, more preferably at least 90% identity to a) or b) or c) providedthat the intracellular signaling activity is maintained; and

(f) the amino acid sequence under (a) or (b) or (c) wherein at most 10,preferably at most 8, more preferably at most 5 amino acid residues aredeleted, added or substituted, provided that the signaling activity, forexample nuclear translocation, is maintained.(g) the consensus sequence VxxxxVQRxAxxELIxVxxxLxPxxxxxKRxRS (SEQ ID NO:10) wherein x is any amino acid residue;(h) the consensus sequence VxxxxxQxxAxxELIxVxxxLxPxxxxxKRKRS (SEQ ID NO:11) wherein x is any amino acid residue;(i) the consensus sequence Vxxxx[V/I]Q [R/H] [Q/K]A[F/V/I][N/H]ELI[R/Q]Vx[H/A] [Q/E]L[L/S]P[E/A][S/A] [S/A] [L/K]xxKRKRS (SEQ IDNO: 12) wherein x is any amino acid residue;

The amino acid sequence under (a) represents a truncated murine INFysequence, the sequence under (b) is a human homologue and the sequenceunder (c) is the rat homolog. Preferably, an analog of the inventioncomprises a stretch of at least 10, preferably at least 15, morepreferably at least 20 contiguous amino acids of the sequence of (a) or(b) or (c). In one embodiment, said stretch comprises the N-terminalsequence of the sequence under (a) or (b) or (c). In another embodiment,it comprises the C-terminal sequence of the sequence under (a) or (b) or(c), preferably at least the last 15 amino acids. In yet anotherembodiment, the stretch comprises an internal sequence of the sequenceunder (a) or (b) or (c). Exemplary sequences are LLPESSLRKRKRSR, (SEQ IDNO: 13) KFEVNNPQVQRQ, (SEQ ID NO: 14) QAFNELIRVVHQLL, (SEQ ID NO: 15)MAELSPAAKTGKRTRSQ, (SEQ ID NO: 16), YSVTDLNVQRKAI, (SEQ ID NO: 17),KAIHELIQVMAELS (SEQ ID NO: 18).

The skilled person will understand that variants with one or more aminoacid modifications to the sequences under (a) or (b) or (c) are alsowithin the scope of the invention. the amino acid sequence under (a) or(b) or (c) wherein at most 10, preferably at most 8, more preferably atmost 5 amino acid residues are deleted, added or substituted, providedthat the signaling activity, for example nuclear translocation, ismaintained.

Alignment of the human and murine INFγ sequences shows that 15 out ofthe 36 residues (41%) are identical and 24 out of the 36 residues (66%)are positively charged. The identity match is done according to clustalW alignment software.

(SEQ ID NO: 19)huInterferon gamma 4 YSVTDLN VQR K AIHE L I Q V MAELS PAAKTG KRTRSQ 39                     + V +   VQR + A +E L I + V + +L  P +    KR RS+(SEQ ID NO: 20)MuInterferon gamma 3 FEVNNPQ VQRQ  AFNE L I  RV VHQLL PESS LRKRK RS R 38The line in between the sequences indicates the conserved residues,yielding a consensus sequence recited under (g) herein above. Alignmentof the human, rat and mouse interferon gamma can provide the consensussequences under (h) and (i). In one embodiment, an analog comprises asignaling moiety according to the consensus sequence (g), (h) or (i)mentioned above. Other useful sequences include an amino acid sequencesshowing at least 70%, preferably at least 80%, more preferably at least90%, most preferably at least 95% identity to (a) or (b) or (c) providedthat the intracellular signaling activity is maintained.

In a specific aspect, the analog comprises a signalling moiety accordingto the consensus sequence:

(SEQ ID NO: 61)Xaa¹Xaa²Xaa³Xaa⁴ Val Xaa⁵ Xaa⁶ Xaa⁷ Xaa⁸ Xaa⁹ Gln Xaa¹⁰ Xaa¹¹ Ala Xaa¹² Xaa¹³ GluLeu Ile Xaa¹⁴ Val Xaa¹⁵ Xaa¹⁶ Xaa¹⁷ Leu Xaa¹⁸ Pro Xaa¹⁹ Xaa²⁰ Xaa²¹ Xaa²² Xaa²³LysArg Lys Arg Ser Xaa²⁴ Xaa²⁵,Xaa¹, Xaa², Xaa³ Xaa⁴ Xaa⁶ Xaa¹¹, Xaa¹³, Xaa¹⁴, Xaa¹⁶ Xaa¹⁷, Xaa¹⁸ Xaa¹⁹Xaa²⁰ Xaa²¹ Xaa²² Xaa²³ Xaa²⁴ and Xaa²⁵ is any amino acid residueXaa⁵ is a polar, uncharged residue such as Asn or ThrXaa⁷ is a non-polar, hydrophobic residue such as Pro or LeuXaa⁸ is a polar, uncharged residue such as Gln or AsnXaa⁹ is a non-polar hydrophobic residue such as Val, Ile or LeuXaa¹⁰ is a polar, basic residue such as Arg, His or LysXaa¹² is a non-polar hydrophobic residue such as Phe, Val or IleXaa¹⁵ is a non-polar hydrophobic residue such as Val, Ile, Met

Preferably, an analog comprises as signaling moiety a sequencecorresponding to residues 95-133 in murine IFNγ or residues 95-134 inhuman IFNγ. This sequence has antiviral activity (Mujtaba et al. 2006,Clinical and Vaccine Immunology, Vol. 13, No. 8, p. 944-952).

The signaling activity of an analog can be readily determined by methodsknown in the art. For example, the capacity of the analog to inducenuclear translocation of STAT1α when taken up intracellularly by amurine macrophage cell line can be determined as described bySubramaniam et al. Signaling activity can also be assessed by complexformation with the transcription factor Stat1alpha and the nuclearimporter of Stat1alpha, the importin-alpha analog NPI-1. See inparticular Subramaniam et al. (2001, J. Interferon Cytokine Res.21(11):951-959). Other suitable in vitro assays include NO-production inmurine macrophages such as the RAW cell line. In one embodiment, theanalog shows at least 30%, preferably at least 50%, more preferably atleast 75% of the (in vitro) activity of its full length interferoncounterpart. Cell specific targeting can significantly enhance the invivo efficacy of an analog showing relatively low activity in vitro.

As mentioned herein above, an analog of the present invention ischaracterized by the absence of a functional IFN-receptor binding domainand the presence of a targeting domain capable of binding to analternative receptor which can mediate intracellular uptake of theanalog, for example a receptor which enters the endocytic pathway uponligand binding and/or as part of constant receptor turn-over. As will beunderstood, the alternative or “secondary” receptor to be targetedshould have some degree of cell type specific expression. Ubiquitouslyexpressed receptors are less suitable candidates. Furthermore, thereceptor must be expressed on a cell which is responsive to theintracellular signaling moiety, e.g. in case of an IFNγ analog it mustcontain an IFNγ-responsive element.

Preferably, the targeting domain can bind to a receptor that is specificfor fibroblast and fibroblast-like cells, such as for examplemyofibroblasts, portal fibroblasts, mesangial cells, interstitialfibroblasts, alveolar fibroblasts or stromal cells. Also envisaged arereceptors expressed on tumor cells. An IFNγ-analog can induce apoptosisin tumor cells, which can be specifically targeted via a secondaryreceptor expressed on tumor cells. In one embodiment, the receptor isselected from the group consisting of the PDGF receptors, collagen typeVI receptor, cytokine receptors including TGF receptor, TNFα receptorand the IL1β receptor, Insulin growth factor receptors, VEGF receptorsand chemokine receptors (e.g. CXCR4). Very good results were obtainedwhen the analog was targeted to the PDGF receptor, preferably the PDGF-βreceptor. Other suitable targeting domains include cell adhesionpeptides, such as the tripeptide Arg-Gly-Asp (RGD) which was originallyidentified as the sequence within fibronectin that mediates cellattachment. The RGD motif has now been found in numerous other proteinsand supports cell adhesion in many, but not all, of these. Theintegrins, a family of cell-surface proteins, act as receptors for celladhesion molecules. A subset of the integrins recognize the RGD motifwithin their ligands, the binding of which mediates both cell-substratumand cell-cell interactions. RGD-containing analogs may be used astherapeutic agents for the treatment of diseases such as thrombosis andcancer.

In a specific aspect, the targeting domain targets can bind to areceptor for an oligosaccharide or a glycoprotein, preferably theasialoglycoprotein (ASGP) receptor or the mannose receptor (CD 206). Forexample, the targeting domain is an oligosaccharide, preferably mannoseor lactose, conjugated to a carrier molecule. In another embodiment,mannose-6-phosphate (M6P) or a derivative thereof can be used astargeting domain for the M6P/IGF II receptor. See for example EP1117443.

The targeting domain is a preferably a proteinaceous substance (peptide)such that the analog as a whole is a fusion polypeptide which can bereadily produced e.g. recombinantly. However, non-proteinaceous othertypes of targeting domains are also envisaged such as saccharides,lipids, nucleic acids, synthetic molecules, and the like.

In one aspect, the targeting domain comprises at least one cyclicpeptide portion. Peptides can be cyclised by various means, includingcysteine-disulfide or lanthionine bridge formation. Cyclic peptidesuseful for targeting an analog of the invention to a desired cell typeare described in the art. For example, EP1117443 discloses a cyclicpeptide comprising at least one sequence encoding a cell receptorrecognising peptide (RPR). In one embodiment, an analog of the inventioncomprises in its (cyclised or linear) targeting domain at least an aminoacid sequence selected from the group consisting of RGD, KPT, SRN, NLIand LID.

A preferred embodiment relates to analogs having a targeting domainwhich comprises multiple receptor binding sequences, preferably in theform of at least one tandem repeat of a (cyclic) peptide portion. Thetandem repeat may comprise two cyclic peptide portions, preferablyidentical cyclic peptide portions, connected via a linker of 1 to 5amino acids. This is of particular advantage for targeting a receptorwhich is active as a dimer, such as the PDGF, TGFβ or IL-10 receptor.

In one embodiment, the targeting domain comprises one, preferably two,copie(s) of the amino acid sequence X₁SRNLIDX₂, (SEQ ID NO: 21) whereinX_(I) and X₂ denote moieties which together can form a (peptidic) bondsuch that a cyclic structure is formed wherein the sequence SRNLID (SEQID NO: 36) is part of the ring. The two copies are preferably spaced bya linker sequence of 1 to 7 amino acids. Preferably, X_(I) and X₂ areCys residues. The inventors found that a targeting domain comprising theamino acid sequence CSRNLIDC(SEQ ID NO:46)-linker-CSRNLIDCS (SEQ ID NO:62), wherein the linker is an amino acid sequence of 1 to 7, like 1, 2,3, 4, 5, 6 or 7, amino acid residues, is very effective in binding tothe dimeric PDGFβ receptor. For example, they performed a comparativeexperiment wherein two cyclic peptides capable of binding to thePDGF-receptor were linked to each other via a spacer consisting of 6amino acids whereas in another construct these two cyclic peptides werecoupled via a spacer of 11 amino acids. In both constructs the spacercontained a lysine residue to allow coupling of a drug or tracer. Bothbicyclic constructs were labeled with FITC and added to 3T3 cellcultures, expressing the PDGF-receptor and incubated for 2 hrs toexamine the binding. Following incubation and staining with an antibodyagainst FITC, immunohistochemical data showed significant binding of theconstruct comprising the spacer of 6 amino acids, whereas only lowbinding was observed with the construct containing the longer spacer of11 amino acids.

In one embodiment, the linker may consist of 4 or 5 amino acid residues,preferably selected from the group of Gly, Ala, Ser and Thr residues,more preferably at least 3 of them being a glycine residue. Preferredlinkers consist of the sequences KGSGG (SEQ ID NO:37) and KGSGSGG (SEQID NO:38). As a specific example, the targeting domain comprises orconsists of the amino acid sequence CSRNLIDCKGSGGCSRNLIDCS (SEQ ID NO:39), or CSRNLIDCKGSGSGGCSRNLIDCS (SEQ ID NO: 40). In another embodiment,the linker consists of 5 or 6 amino acid residues, preferably selectedfrom the group of Asp, Lys, Gly, Ala, Ser and Thr residues. Good resultswere obtained with a linker of 4 to 7 residues, at least 4, preferablyat least 5, being a glycine residue. Other suitable linkers include asequence of 4 to 7 residues, the residues being selected from Gly andAsp residues, e.g. [G_(n) D_(m)] wherein n+m is from 4 to 7, wherein n≧4and M an integer between 0 and 3. In a specific embodiment, thetargeting domain comprises or consists of the amino acid sequenceCSRNLIDC[GnD m]CSRNLIDC, (SEQ ID NO: 63), wherein n+m is from 4 to 7,wherein n≧4 and M an integer between 0 and 3. For example, the targetingdomain consists of or comprises the sequence CSRNLIDCGGGDGGCSRNLIDC,(SEQ ID NO: 41), CSRNLIDCGGDGGCSRNLIDC, (SEQ ID NO: 42),CSRNLIDCGDDGGCSRNLIDC (SEQ ID NO: 43), or CSRNLIDCGGGGGGCSRNLIDC (SEQ IDNO: 44).

The targeting domain may be attached to the signalling domain by anymeans, for instance by a linker or spacer sequence. Suitable linkersequences are typically up to 15, preferably up to 10 amino acidresidues in length. Polyalanine linkers may be used. For instance,provided herein is an interferon gamma analog, consisting of thesequence

CSRNLIDCKGSGGCSRNLIDCSAAAAKFEVNNPQVQRQAFNELIRVVHQLLPESS LRKRKRSR, (SEQID NO: 30),

CSRNLIDCKGSGSGGCSRNLIDCSAAAAKFEVNNPQVQRQAFNELIRVVHQLLPE SSLRKRKRSR (SEQID NO: 31), or

CSRNLIDCGGGDGGCSRNLIDCSAAAKFEVNNPQVQRQAFNELIRVVHQLLPESS LRKRKRSR (SEQ IDNO: 32).

The targeting domain may also be attached to the signaling domain via acarrier molecule, e.g. albumin. This is especially advantageous if thetargeting domain is a non-proteinaceous substance e.g. anoligosaccharide such as mannose or lactose. In one embodiment, thecarrier molecule comprises free reactive groups like hydroxyl, amineand/or sulphate. The size of the carrier is preferably an endogenousplasma protein, like albumin, lactoferrin or fibronectin.

The invention also relates to a conjugate comprising a compound ofinterest, e.g. a biologically active molecule, conjugated to a bicyclicPDGF-targeting domain, said targeting domain comprising two copies ofthe amino acid sequence X₁SRNLIDX₂, (SEQ ID NO: 21), wherein X₁ and X₂denote moieties which together can form a bond, like a peptidic ordisulphide bond, such that a bicyclic structure is formed wherein thesequence SRNLID (SEQ ID NO: 36), is part of each ring. Preferably, X₁and X₂ are Cys residues allowing for cyclisation via disulphide bondformation. A conjugate according to the invention is characterized inthat the two copies are spaced by a linker sequence of 2 to 7 aminoacids. It was surprisingly found that this specific spacer length allowsfor highly efficient targeting of the PDGF receptor, which is active asa dimer. Thus, also provided is a conjugate comprising a compound ofinterest conjugated to a targeting domain, said targeting domaincomprising the amino acid sequence X₁SRNLIDX₂(SEQ IDNO:21)-linker-X₃SRNLIDX₄, (SEQ ID NO: 21), wherein the pair of X₁ and X₂and the pair of X₃ and X₄ can form a (peptidic) bond such that abicyclic structure is formed wherein the sequences SRNLID (SEQ ID NO:36), are part of a ring, and wherein the linker is an amino acidsequence consisting of 2 to 7 amino acid residues. As discussed hereinabove, the linker may consist of 4 or 5 amino acid residues. They can beselected from the group of Gly, Ala, Ser, Asp, Lys and Thr residues,preferably at least 3 of them being a glycine residue. Preferred linkersconsist of the sequences KGSGG (SEQ ID NO: 37) and KGSGSGG (SEQ IDNO:38). As a specific example, the targeting domain comprises orconsists of the amino acid sequence CSRNLIDCKGSGGCSRNLIDCS (SEQ ID NO:39), or CSRNLIDCKGSGSGGCSRNLIDCS (SEQ ID NO: 40). In another embodiment,the linker consists of 5 or 6 amino acid residues, preferably selectedfrom the group of Asp, Gly, Ala, Ser and Thr residues. Good results wereobtained with a linker of 4 to 7 residues, at least 4, preferably atleast 5, being a glycine residue. Other suitable linkers include asequence of 4 to 7 residues, the residues being selected from Gly andAsp residues, e.g. [GnD m] wherein n+m is from 4 to 7, wherein n≧4 and Man integer between 0 and 3. In a specific embodiment, the targetingdomain comprises or consists of the amino acid sequence CSRNLIDC[GnDm]CSRNLIDC, (SEQ ID NO: 63), wherein n+m is from 4 to 7, wherein n≧4 and Man integer between 0 and 3. For example, the targeting domain consistsof or comprises the sequence

CSRNLIDCGGGDGGCSRNLIDC, (SEQ ID NO: 41),

CSRNLIDCGGDGGCSRNLIDC, (SEQ ID NO: 42),

CSRNLIDCGDDGGCSRNLIDC (SEQ ID NO: 43), or

CSRNLIDCGGGGGGCSRNLIDC (SEQ ID NO: 44).

The use of the sequence SRNLID (SEQ ID NO: 36), as cell targeting domainis known in the art. W000/23113 discloses the conjugation of a cyclicpeptide comprising a receptor recognizing peptide (RRP) to a carriermolecule being larger than 5000 Dalton, for instance serum albumin. Anexemplary RRP is the PDGF receptor binding sequence XSRNLIDCX, (SEQ IDNO: 45), wherein X denotes the location of cyclisation. It is taughtthat, within the cyclic peptide structure, multiple RRP sequences may bepresent. It is also disclosed to attach more than one (e.g. 5-15 cyclicpeptides) to a carrier molecule. Hagens et al. (2007) PharmaceuticalRes. Vol. 24, pp. 566-574, show the delivery of cyclic peptide CSRNLIDC(SEQ ID NO: 46), conjugated to albumin to hepatic stellate cells. Thus,the prior art conjugates all rely on attaching RRPs to a carriermolecule. The construction of conjugate of the invention, wherein thecyclic moieties are not attached to a carrier molecule but insteadthereof placed in a tandem motif with a specific spacer length of 2 to 7amino acids is not taught or suggested in the art. It was found thatthis well defined bicyclic structure enhances conjugate binding to thedimeric PDGF receptor as compared to a conjugate according toW000/23113, wherein the number and spatial orientation of the multiplereceptor binding sequences attached to the carrier are randomized andmuch less controlled. The tandem configuration fixing the distancebetween the two cyclic moieties in a conjugate of the invention isdesigned to interact optimally with the dimeric PDGF-receptor.Furthermore, the presence of the relatively large carrier molecule maydecrease receptor interactions by steric hindrance.

The bicyclic peptide can be prepared chemically or through recombinanttechniques which provides production methods that are very favourable tothe pharmaceutical industry. In addition, the bicyclic structure can bedirectly attached to a chemical entity (drug or tracer), polymer (egPolyethylene glycol, PEG), small peptide or protein yielding acell-specific low molecular weight compound that can easily penetrateinto extravascular tissues. In particular for tumor targeting purposes,this tissue penetration may be very relevant.

Very good results were achieved with a linker consisting of 3 to 5 aminoacid residues. In one embodiment, the conjugate comprises the amino acidsequence CSRNLIDC(SEQ ID NO:46)-linker-CSRNLIDCS(SEQ ID NO: 62), (BiPPB)wherein the linker is an amino acid sequence of 2 to 7, preferably 3 to5, amino acid residues. This bicyclic peptide is suitably used as lowmolecular weight targeting ligand with high affinity to the PDG Freceptor (PDGF-R). The peptide can be prepared either by chemical orrecombinant synthesis. The linker may comprise one or more amino acidresidues with a reactive side chain that can be used for covalentattachment of a compound of interest, like a detectable label, a drugand/or diagnostic. Suitable reactive amino acids include lysine, serineand threonine, arginine, histidine, aspartic acid, glutamic acid,cysteine, asparagine, glutamine, tyrosine, methionine and tryptophan.

The biologically active moiety may have any type of useful biologicalactivity, including cytokine, chemokine, or prostaglandin activity. Itcan be of proteinaceous or non-proteinaceous nature. For instance, themoiety is selected from the group selected from drugs, cytokines,chemokines, hormones, prostaglandins, and the like. Specific examplesinclude PGE2, 15d-PGJ2, IL-10, IFNγ, truncated IFNγ. Proteinaceousmoieties may conveniently be attached to the PDGF-R specific targetingdomain by genetic fusion, at either the N- or C-terminus. In oneembodiment, it is conjugated to the N-terminus.

An analog or conjugate according to the invention may be coupled to acore and/or carrier or delivery molecule by methods known in the art.Suitable cores or carriers include dendrimers, liposomes, and natural,synthetic or semi-synthetic polymers (branched or linear). Dendrimershave successfully proved themselves as useful additives in differentroutes of drug administration because they can render drugs greaterwater-solubility, bioavailability, and biocompatibility. See Chen etal., Journal of Pharmaceutical Sciences, Vol. 97 Issue 1, μg. 123-143.As an example, the invention provides an IFNγ-analog conjugated to adendrimer or to a liposome. The liposome may contain (anti-cancer) drug.

In one embodiment, an interferon analog or PDGFR-targeted conjugateaccording to the invention is modified by conventional means to improveits pharmacological properties, like enhancing the efficacy and/orstability. In one embodiment, it is modified in order to enhance thehalf life by the attachment of at least one non antigenic polymer, forinstance by a polymer selected from the group consisting of polyethyleneglycols (PEGs) and derivatives thereof. PEGylation is routinely achievedby incubation of a reactive derivative of PEG with the targetmacromolecule. The covalent attachment of PEG to a drug or therapeuticprotein can “mask” the agent from the host's immune system (reducedimmunogenicity and antigenicity), increase the hydrodynamic size (sizein solution) of the agent which prolongs its circulatory time byreducing renal clearance.

A further aspect of the invention relates to an isolated nucleic acidsequence encoding a proteinaceous interferon analog according to theinvention or encoding a proteinaceous PDGFR-targeted conjugate asdescribed herein above. The skilled person will be able to design andconstruct a suitable nucleic acid sequence e.g. a fusion construct thatencodes both the targeting moiety and the biologically active moietyusing standard recombinant DNA technology. The isolated nucleic acidsequence may be part of an expression vector, for example a vectordesigned for recombinant protein production in a bacterial or mammalianhost cell. Also provided is a host cell comprising a nucleic acidsequence or a vector according to the invention, preferably wherein saidhost cell is a bacterial or mammalian host cell. An analog orPDGF-targeted conjugate disclosed herein has improved properties withrespect to its distribution within the body. More specifically, itallows directing a biologically active compound of interest to a cell ofinterest while maintaining the biological activity of that particularcompound. To attain cell-specificity, these mediators are equipped withan address label that will increase their concentration around relevanttarget receptors in diseased tissue. A further embodiment thereforerelates to a pharmaceutical composition comprising an IFN analog or aPDGF targeted conjugate and a pharmaceutically acceptable carrier. Aspecific aspect relates to a pharmaceutical composition comprising atargeted IFNγ analog, preferably a PDGF-targeted IFNγ analog showingreduced side-effects as compared to non-targeted IFNγ. An exemplarypharmaceutical composition contains a peptide comprising or consistingof the sequence CSRNLIDCKGSGGCSRNLIDCSAAAAKFEVNNPQVQRQAFNELIRVVHQLLPESSLRKRKRSR (SEQ ID NO: 30), orCSRNLIDCKGSGSGGCSRNLIDCSAAAAKFEVNNPQVQRQAFNELIRVVH QLLPESSLRKRKRSR (SEQID NO: 31). Another exemplary pharmaceutical composition comprises aconjugate comprising a biologically active molecule conjugated to atargeting domain, said targeting domain comprising the amino acidsequence CSRNLIDC(SEQ ID NO:46)-linker-CSRNLIDCS, (SEQ ID NO: 62),wherein the linker is an amino acid sequence of 2 to 7, preferably 3 to5, amino acid residues.

Also provided is the use of an IFN (γ) analog according to the inventionfor the manufacture of a medicament for the therapeutic or prophylactictreatment of a disease selected from cancer, viral diseases, fibroticdisease, sclerotic disease and chronic or acute inflammatory diseases.Exemplary diseases include glomerulosclerosis, interstitial fibrosis,lung fibrosis (idiopathic pulmonary fibrosis), atherosclerosis,rheumatoid arthritis, Crohns disease, colitis ulcerosa,glomerulonephritis and sepsis.

It is known that INF_(Y) also has antiviral activity. Since thetruncated form of INF_(Y) is shown to be bioactive and found to displayall activities of native INF_(Y) provided a homing device is attached tothe molecule, an analog of the invention is endowed with antiviralactivity as well. Exemplary diseases included infections caused byinfluenza virus or human respiratory syncytial virus (RSV). RSV is avirus that causes respiratory tract infections. It is the major cause oflower respiratory tract infection and hospital visits during infancy andchildhood. Sometimes an infant can become symptomatically infected morethan once, even within a single RSV season. In the United States, 60% ofinfants are infected during their first RSV season, and nearly allchildren will have been infected with the virus by 2-3 years of age. Ofthose infected with RSV, 2-3% will develop bronchiolitis, necessitatinghospitalization. Severe RSV infections have increasingly been foundamong elderly patients. There is no vaccine. Treatment is limited tosupportive care, including oxygen. In temperate climates there is anannual epidemic during the winter months. In tropical climates,infection is most common during the rainy season.

The invention hence also relates to a method for therapeutic orprophylactic treatment of a disease selected from cancer, viral disease,fibrotic disease, sclerotic disease and chronic or acute inflammatorydiseases such as glomerulosclerosis, interstitial fibrosis, lungfibrosis, atherosclerosis, rheumatoid arthritis, Crohns disease, colitisulcerosa, glomerulonephritis and sepsis, comprising providing to asubject in need thereof a therapeutically effective dose of an IFNanalog according to the invention. The disease may be a liver disease,preferably a chronic liver disease such as liver cirrhosis. The personskilled in the art will adjust the dosage to be applied to the manner ofapplication, size, weight, state of health etc of the subject to whichadministration is to occur. Administration can occur in any manner knownper se for administration of a medicament. An IFNγ analog according tothe invention is advantageously used in a medicinal composition(therapeutic or prophylactic) in a form for intrapulmonary delivery,e.g. by intranasal administration or inhalation. Also provided is aninhalation device comprising an IFNγ analog as active ingredient.

LEGEND TO THE FIGURES

FIG. 1: cloning of mimetic IFNγ from the mouse. Splenocytes werestimulated with PHA and RNA was isolated. RT and PCR using specificprimers yielded a PCR product of the expected size. Expression of theconstruct in BL21 cells and Western blotting confirmed production of themimetic.

FIG. 2: Recombinant mimetic IFNγ shows anti-fibrotic effects in mouse3T3 fibroblasts. Cells are stained for α-Smooth muscle actin which is afibrosis marker.

FIG. 3: Construction of pET39b-BiPPB encoding a bicyclic PDGF-receptortargeting domain.

FIG. 4: Construction of pET39b-BiPPB-IFNgamma encoding a PDGFR-targetedIFNγ conjugate.

FIG. 5: Construction of pET39b-BiPPB-mimetic IFNγ.

FIG. 6: Dot Blot analysis of IFNγ-BiPPB and Mimetic IFNγ-BiPPB showingthe coupling of pPB-peptide to INF_(Y).

FIG. 7: Binding study in human hepatic stellate cell line LX2 cells.Cells were stained for PDGFR-binding peptide. Staining indicates thebinding of pPB-containing constructs to the target cells.

FIG. 8: Blood count analysis to study side effects of the treatment inacute liver injury mouse model. WBC=white blood cell count.LYM=lymphocytes. PLT=platelet count. RBC=red blood cell count. Fordetails see Example 4.

FIG. 9: Real Time PCR analysis of the liver fibrosis markers MMP13 andTIMP1. For details see Example 4.

FIG. 10: Effect of mimetic IFNγ-PEG-BiPPB in the acute CCl4-inducedliver-injury mouse model. Quantitation of a-SMA (A), collagen-I (B) anddesmin (C)—staining of liver sections obtained from olive oil-treatednormal mice and CCl4-treated mice receiving either IFNγ, mimetic IFNγ,mimetic IFNγ-PEG, mimetic IFNγ-PEG-BiPPB (5 μg/mice) or PBS alone.Quantitation was done using computerized Cell-D imaging software. Datarepresent the mean±SEM from 5 mice per group. #P<0.05 versus PBS-treatedolive oil group; *P<0.05, **P<0.01 versus PBS treated-CCl4 group. Fordetails see Example 6.

FIG. 11: Real Time PCR analysis of markers for liver fibrosis in mice, 3days after CCl4 administration or olive oil. Mice were treated withdifferent compounds as indicated and Collagen 1a1 (A), a-SMA (B), Desmin(C) and TIMP1 (D) mRNA levels were measured. For details see Example 6.

FIG. 12: Blood count analysis in whole blood of mice, 3 days after CCl4administration or olive oil. Mice were treated with different compoundsas indicated and Platelet count (PLT: fig A), white blood cell count(WBC, fig B) and lymphocyte count (C) were measured using a coultercounter. For details see Example 6.

EXPERIMENTAL SECTION

Liver Fibrosis is characterized by the excessive accumulation of theextracellular matrix components that leads to hepatic scars. To date, nosuccessful therapy is available for the treatment of liver fibrosis.Hepatic stellate cells (HSCs) and fibroblasts are the key effector cellsinvolved in the progression of the disease, which are activated bycrucial growth factors like Platelet derived growth factor (PDGF) andtransforming growth factor-beta (TGFβ). Interferon gamma (IFNγ) has beenshown to have various beneficial effects in-vitro and in-vivo duringliver fibrosis. However, IFNγ displays a strict species specificity andhas a short circulating half life which limits its potential clinicaluse. Moreover, INFγ has serious adverse effects on the immune system, onendothelial cells and on the Central Nervous System (e.g. causingdepressions) that all lead to frequent withdrawal of patients fromclinical trials and consequently to failure of these trails. Tocircumvent these drawbacks, the present inventors produced a stablepeptide mimetic of IFNγ that lacks the extracellular receptorrecognition site and contains a signaling moiety which interactsdirectly at the downstream IFNγ signaling cascade, thereby retaining theprospective functions of IFNγ.

To increase the specificity of IFNγ and IFNγ mimetic peptide, IFNγ andmimetic peptide fused to BiPPB (Bicyclic peptide against thePDGF-beta-receptor) were been generated, since PDGF receptor expressionis highly up-regulated during liver injury particularly in HSCs.

Example 1 Cloning of Mimetic IFNγ

Mouse splenocytes were isolated from fresh spleens and were cultured inthe presence of PHA (Phytohemagglutinin) to stimulate cytokineproduction. After 24 hrs of stimulation, RNA was isolated and cDNA wassynthesised using gene specific reverse primer followed by PCRamplification by phusion DNA polymerase using mimetic IFNγ specificforward and reverse primers. The obtained fragment was cloned in pET42a(prokaryotic expression vector) at PshA1/EcoRI site and the positiveclones were checked by restriction digestion analysis. See FIG. 1.

The 5′→3′ nucleotide sequence of the truncated mouse Interferon gamma(NCBI Reference sequence: NM_008337.2) (nt 457-nt 572) is as follows:

457 gcca agtttgaggt caacaaccca caggtccagc gccaagcatt caatgagctcatccgagtgg tccaccagct gttgccggaa tccagcctca ggaagcggaa aaggagtcgc tg 572a (SEQ ID NO:49).

The last nucleotide has been added in order to insert the stop codonbefore cysteine, which is the last amino acid in the sequence. Cysteineis removed from the sequence to provide appropriate folding of thepeptide and also to avoid inappropriate folding due to disulfide bondsin the fusion protein (with BiPPB).

The encoded amino acid sequence is

(SEQ ID NO: 50) AKFEVNNPQVQRQAFNELIRVVHQLLPESSLRKRKRSR**Denotes Stop codon* Denotes Stop codon

Cloning of IFNγ-BiPPB and Mimetic IFNγ-BiPPB

A nucleic acid sequence encoding the bicyclic PDGF targeting domainBiPPB was generated by amplification of two fragments using 4 primers (2for each fragment) and then ligated using inbuilt Bam HI restrictionsite and was then cloned in pET39b vector at ScaI/NotI site. IFNγ andMimetic IFNγ was PCR amplified using peptide fusion primer (forward) andIFNγ or mimetic IFNγ reverse primer. The amplified fragment was digestedand ligated in pET39b-BiPPB vector at NotI/XhoI site and the positiveclones were checked by restriction digestion analysis. See FIGS. 4 and5. The sequences of all the constructs were further confirmed byautomated DNA sequencing.

BiPPB

Nucleotide sequence for BiPPB: (SEQ ID NO: 51)tgt tct aga aac ctc atc gat tgt aag gga tcc gga ggt tgt tca cgt aat cta ata gat tgt tcaAmino acid sequence for BiPPB:  (SEQ ID NO: 52)CSRNLIDCKGSGGCSRNLIDCS (see also FIG. 3)Interferon Gamma (Full Length)

Nucleotide sequence: mouse Interferon gamma (NCBI Reference sequence: NM_008337.3)(SEQ ID NO: 53)gcggccgca 457 gcca agtttgaggt caacaaccca 481 caggtccagc gccaagcatt caatgagctcatccgagtgg tccaccagct gttgccggaa 541 tccagcctca ggaagcggaa aaggagtcg 569 ataaAmino acid sequence for Mouse IFNgamma (SEQ ID NO: 54)MNATHCILALQLFLMAVSGCYCHGTVIESLESLNNYFNSSGIDVEEKSLFLDIWRNWQKDGDMKILQSQIISFYLRLFEVLKDNQAISNNISVIESHLITTFFSNSKAKKDAFMSIAKFEVNNPQVQRQAFNELIRVVHQLLPESSLRKRKRSRCNucleotide sequence of the fused protein (BiPPB-IFN gamma)(SEQ ID NO: 55)tgt tct aga aac ctc atc gat tgt aag gga tcc gga ggt tgt tca cgt aat cta ata gat tgt tcagcggccgca Interferon gamma sequence (NM_008337.3).Amino acid sequence for BiPPB-IFNgamma (SEQ ID NO: 56)CSRNLIDCKGSGSGGCSRNLIDCS AAAMNATHCILALQLFLMAVSGCYCHGTVIESLESLNNYFNSSGIDVEEKSLFLDIWRNWQKDGDMKILQSQIISFYLRLFEVLKDNQAISNNISVIESHLITTFFSNSKAKKDAFMSIAKFEVNNPQVQRQAFNELIRVVHQLL PESSLRKRKRSRCItalics denotes BiPPB; normal text denotes IFNgamma mimetic; Bolddenotes linker or spacer. See also FIG. 4.Mimetic Interferon Gamma Fused to BiPPB

Nucleotide sequence of the fused protein (BiPPB-IFN gamma mimetic)(SEQ ID NO: 57)tgt tct aga aac ctc atc gat tgt aag gga tcc gga ggt tgt tca cgt aat cta ata gat tgt tcagcggccgca 457 gcca agtttgaggt caacaaccca caggtccagc gccaagcatt caatgagctc atccgagtggtccaccagct gttgccggaa tccagcctca ggaagcggaa aaggagtcg 569 ataaAmino acid sequence for BiPPB-IFNgamma mimetic (SEQ ID NO: 58)CSRNLIDCKGSGSGGCSRNLIDCS AAA AKFEVNNPQVQRQAFNELIRVVHQLLPESSLRKRKRSR*Italics denotes BiPPB; normal text denotes IFNgamma mimetic; Bolddenotes: linker or spacer. See also FIG. 5.

The nucleic acids were then transformed into BL21 cells (E. coli) forthe expression using IPTG induction. The expressed protein was analysedby SDS-PAGE and Western blot analysis or Dot Blot analysis usinganti-IFNγ antibody and/or anti-PPB antibody (see FIG. 6). The expressedprotein (with His Tag) was then purified under native conditions throughNi-NTA column chromatography. The tags were proteolytically cleaved andfurther purified using sepharose column chromatography.

Example 2 Anti-Fibrotic Effects of Cleaved and Active Mimetic IFNγ

We evaluated the anti-fibrotic effects of cleaved IFNγ mimetic peptidein mouse NIH3T3 fibroblasts as assessed by immuno-cytochemistry (α-SMAstaining). Briefly, 3T3 fibroblasts were seeded in 24 well plates at thedensity of 6×10⁴ cells/well, After 24 hrs, cells were starved in 0.5%FBS containing medium for overnight. Thereafter, cells were incubated instarvation medium with different compounds (PDGF 50 ng/ml, TGFβ 10ng/ml, mimetic IFNγ 1 μg/ml, 50 ng/ml PDGF+1 μg/ml mimetic IFNγ and 10ng/ml TGFβ+1 μg/ml mimetic IFNγ). After 48 hrs of incubation, cells werewashed, fixed with ethanol:acetone (1:1) and stained for α-SMA (markerof activated fibroblasts).

Example 3 Binding Study of BiPPB and Mimetic IFNγ-BiPPB in Human LX₂Cells

We determined the binding of BiPPB and mimetic-BiPPB in LX₂ cells (humanHSCs). Briefly, LX₂ cells were plated in 48 well plates at the celldensity of 3×10⁴ cells/well. After 24 hrs, cells were starved in medium(-FBS) for overnight. Then, cells were incubated with differentcompounds (BiPPB, PPB-HSA and BiPPB-Mimetic IFNγ) for 2 hrs at roomtemperature for binding. After binding, cells were extensively washedwith PBS, fixed with ethanol:acetone (1:1) and stained for PPB. Resultsare presented in FIG. 7.

Example 4 In-Vivo Effect Study in Acute CCl₄-Induced Liver Injury inMice

Recombinant IFNγ, mimetic IFNγ, recombinant fusion protein IFNγ-BiPPBand recombinant fusion protein mimetic IFNγ-BiPPB were tested foranti-fibrotic effects in acute CCL₄-induced liver injury mouse model. Atday 1, the animals were given a single intra-peritoneal dose (1 ml/kg)of carbon tetrachloride (CCl₄) in olive oil or olive oil (controls n=6).After 24 hrs of CCl₄ injection, at day 2 and 3, animals were treatedeither with PBS (n=6), 50,000 U/mice of IFNγ (n=6), 50,000 U/mice ofmimetic IFNγ (n=5), 50,000 U/mice of IFNγ-BiPPB (n=6), 50,000 U/mice ofMimetic IFNγ-BiPPB (n=6). Thereafter, at day 4, animals were sacrificedand blood counts were performed and anti-fibrotic effects (See HemmannS, Graf J, Roderfeld M, Roeb. J Hepatol. 2007 May; 46(5):955-75) wereevaluated using quantitative PCR. Results are presented in FIGS. 8 and9.

The data presented in FIG. 8. demonstrate that mimetic-BiPPB is moreeffective than unmodified INF_(Y) in attenuating the upregulation ofwhite blood cell count (WBC) and lymphocyte count (lym) in whole bloodassociated with CCl₄-induced liver fibrosis (p<0.01). In contrast, thereduction in platelet count (PLT) associated with INF_(Y) treatment(which is a well known adverse effect of INF_(Y)) is less severe whenanimals receive mimetic-INF_(Y)-BiPPB instead of unmodified INF_(Y)(p<0.0025).

The data presented in FIG. 9 show that mimetic-INF_(Y)-Bi-PPB is endowedwith antifibrotic activity: it attenuates CCl₄-induced upregulation ofmatrix metalloproteinases (MMP13). The ratio MMP-13 versus TissueInhibitor of Metalloproteinases (TIPM1) is similar to that obtained withnative INF_(Y), but better than mimetic INF_(Y) (p<0.03), indicatingthat coupling of Bi-PPB to mimetic INF_(Y) is beneficial. Collectivelythe data in FIGS. 8 and 9 show that mimetic INF_(Y)-BiPPB is more potentcompared to native INF_(Y) and has less side effects.

Example 5 Chemical Synthesis of Mimetic Interferon Gamma Conjugates

Mimetic IFNγ-PEG-BiPPB Conjugate:

0.111 μmol Bicyclic PDGFR recognizing peptide (BiPPB, 2223.2 Da,Genosphere Biotechnologies) was coupled with 0.337 μmolmaleimide-PEG-succinimidyl carboxy methyl ester (Mal-PEG-SCM, 2 KDa,Creative PEGworks) for 3 hrs. Thereafter, lysine (0.337 μmol) was addedto block free groups of Mal-PEG-SCM. After 1 hr of reaction, thesynthesized product BiPPB-PEG-MAL (0.112 μmol) was reacted with 0.56μmol of Mimetic IFNγ-ATA (4689 Da, Genosphere Biotechnologies) in thepresence of deacetylating reagent for overnight at room temperature.Finally, the synthesized Mimetic IFNγ-PEG-BiPPB conjugate (8828.2 Da)was extensively dialyzed against PBS using 7 KDa slide-a-lyzer G2dialysis cassettes (Thermo scientific).

Mimetic IFNγ-PEG Conjugate:

0.107 μmol Mimetic IFNγ-ATA (4689 Da) was reacted with 0.321 μmol ofPoly(ethylene glycol)-succinimidyl α-methylbutanoate (mPEG-SMB, 2 KDa,Nektar therapeutics) for 2 hrs and subsequently the product was dialyzedextensively.

Example 6 Effect on Fibrotic Parameters after Intravenous Administrationof mimeticIFNγ-PEG-BiPPB in Acute Liver Injury Mouse Model

Analysis of Fibrotic Parameters at the Protein Level:

Mice were intraperitoneally injected with CCl₄ at day 1 to induce liverinjury. At day 2 and 3, mice were treated with IFNγ (5 ug/dose), mimeticIFNγ-PEG, mimetic IFNγ-PEG-BiPPB (5 μg/dose) or PBS alone. At day 4,animals were sacrificed; livers and different organs were collected forfurther analysis. Liver-sections were fixed with acetone, dried andrehydrated with PBS. Then, the sections were incubated with primaryantibody (collagen, SMA and Desmin) for 1 hr. Thereafter, the sectionswere blocked with 0.03% H₂O₂ for endogenous peroxidase activity for 30min. Subsequently, sections were incubated with secondary antibody HRPconjugated rabbit anti-goat antibody (1:100, DAKO) followed by HRPconjugated goat anti-rabbit antibody (1:100, DAKO) for 30 min. Theperoxidase activity was developed using AEC (Sigma) for 20 min andnuclei were counterstained with hematoxylin (Fluka). The sections weremounted with Kaiser's gelatin and visualized under the light microscope(Olympus). For quantitative analysis, 27 microscopic pictures werecaptured and positively-stained areas were quantified using computerizedOlympus Cell D imaging software. Results are shown in FIG. 10

Analysis of Fibrotic Parameters at the Gene Expression Level:

Total RNA from liver tissues was isolated using RNeasy mini kit (Qiagen)according to the manufacturer's instructions. The RNA concentration wasquantitated by a UV spectrophotometer (NanoDrop Technologies,Wilmington, Del.). Total RNA (1.6 μg) were used for reversetranscription in total volume of 50 μl with the cDNA synthesis kit(Promega). All primers were purchased from Sigma-Genosys (Haverhill,UK). 10 ng of cDNA was used for quantitative real time PCR analysis. Thereactions were performed using SYBR green PCR mix (Applied Biosystems)according to manufacturer's instructions. The samples were analyzed byABI 7900HT sequence detection system (Applied Biosystems). Finally, thethreshold cycle numbers (Ct) were calculated for each gene and relativegene expression was calculated after normalizing for expression of thereference gene GAPDH. Results are shown in FIG. 11. Analysis of adverseeffects is depicted in FIG. 12.

Example 7 Effect on Fibrotic Parameters after Intravenous Administrationof mimeticIFNγ-PEG-BiPPB in Established Advanced Liver Fibrosis MouseModel

Analysis of Fibrotic Parameters at the Protein Level:

Male balb/c mice (20-22 g) were treated with olive oil or increasingdoses of CCl₄ (week 1: 0.5 ml/kg; week 2: 0.8 ml/kg and week 3-8: 1ml/kg prepared in olive oil) twice weekly by intra-peritoneal injectionsfor 8 weeks. In week 7 and 8, mice were treated intravenously with PBS,mimeticIFNγ-PEG or MimeticIFNγ-PEG-BiPPB (5 μg/mice, thrice per week).All mice were sacrificed at week 8; blood and liver samples werecollected for subsequent measurements. The liver sections were stainedfor Collagen I and desmin, CD68, 33D1 and MHC class II. It was foundthat the targeted truncated form of IFNγ (mimIFNγ-biPPB) inducedsubstantial reduction in the fibrotic parameters in this chronic liverfibrosis model in mice; both collagen I and desmin staining wasprofoundly reduced in mimIFNγ-biPPB-treated CCl₄ mice compared tountreated CCl₄-mice. In contrast, treatment with untargeted mimIFNγ,lacking the receptor binding site, induced no effect on these parameterswhile full length mouse IFNγ induced only a small reduction in collagenI and desmin staining. The native (mouse) IFNγ induced infiltration ofinflammatory cells (CD68⁺ macrophages, neutrophils, 33D1⁺ dendriticcells) as well as increased MHCII expression. In contrast, MimIFNγ-BiPPBdid not induce this increased inflammatory response in livers.

Example 8 Truncated IFNγ Analog Targeted to the PDGF Receptor is Activebut Causes Less Side Effects

A targeted conjugate of BiPPB chemically coupled mimetic-IFNγ wassynthesized and characterized using Western blot analyses and for itsanti-fibrotic effects in vitro in mouse 3T3 fibroblasts. In vivo, thetargeted conjugate was examined in 4 days (acute) and 8 weeks (chronic)liver fibrosis models induced with CCl₄ in mice. Several fibroticparameters and infiltration of inflammatory cells were assessed in thelivers using immunohistochemistry and gene expression analysis.

Results:

The successfully synthesized conjugate caused inhibition of collagenexpression in TGFbeta-induced mouse fibroblasts. In vivo, the targetedpeptidomimetic of IFNγ (mimIFNγ-biPPB) induced substantial reduction inthe fibrotic parameters in both acute and chronic liver fibrosis modelsin mice. Treatment with untargeted mimIFNγ, which lacks a receptorbinding domain, showed no effect and unmodified mouse full length IFNγshowed only a moderate reduction. This mouse full length IFNγ inducedinfiltration of inflammatory cells (CD68⁺ macrophages, neutrophils,33D1⁺ dendritic cells) as well as increased MHCII expression. Incontrast, MimIFNγ-BiPPB did not induce an inflammatory response (datanot shown).

Example 9 Study with MimeticIFNγ-PEG-BiPPB in Subcutaneous Tumor Modelin Mice

Materials and Methods

Normal male C57BL/6 and Balb/c mice weighing 20 to 25 g were obtainedfrom Harlan (Zeist, the Netherlands). They were kept at a 12:12-hourlight/dark cycle and received ad libitum normal diet. All experimentalprotocols for animal studies were approved by the Animal EthicsCommittee of the University of Groningen. To induce subcutaneous tumors,C26 cells were cultured in 125-mm³ flasks a day before injection inanimals to keep them in the growth phase. Cells were detached bytrypsanization, and trypsin was removed by centrifugation. The cellpellet was resuspended in PBS. A total of 1×10⁶ cells (B16 and C26cells) suspended in 100 μl of PBS were injected subcutaneously in theflank of Balb/c mice. Tumor growth was followed by measuring tumor sizeusing a digital Vernier caliper. Tumor volume was established using theformula: a×b2/2, where a denotes tumor length and b denotes the tumorwidth. C26 tumors were induced in mice as described. The treatment wasstarted on day 5 when the tumor volume was reached the range of 50 to100 mm³ because this tumor size has been shown as an optimum tumor sizefor the start of the treatment. Animals (n=4 per group) were injectedintravenously with six doses of either vehicle (PBS), mimeticIFNγ-PEG (5μg/dose), mimeticIFNγ-PEG-BiPPB (5 μg/dose) on alternative days underanesthesia (O₂/isoflurane). Tumor size was measured under anesthesia.The animals with C26 tumors were killed on day 20 because no effect ofthe treatment was observed. Animals were killed under gas anesthesia(O₂/isoflurane), and tumors were isolated and fixed in cold isopentanefor cryosections.

4-μm-thick cryostat sections were prepared from snap-frozen tissue andstained for CD31 according to standard immunoperoxidase methods. Weanalyzed CD31 staining (endothelial cell marker) for the determinationof the blood vessel lumen area and blood vessel density in tumorsections of C26 tumors. Results showed significant angiogenesis inuntreated tumors and in tumors of mice treated with mimetic IFNγ, whilemice treated with mimetic IFNγ-BiPPB displayed a strong reduction inangiogenesis in their tumors (data not shown).

Example 10 Effect of PDGF-Receptor Targeted Truncated INF_(Y) onPulmonary Fibrosis

Background and Rationale for INF_(Y)-Based Therapies in IPF Patients:

Idiopathic pulmonary fibrosis (IPF) is a progressive parenchymal lungdisease with a median survival of only 3-5 years followingdiagnosis^(1, 2). Besides IPF also other types of lung fibrosis have apoor prognosis, in particular fibrosing non-specific interstitialpneumonia (NSIP) and end-stage fibrosis of for example severalauto-immune diseases and extrinsic allergic alveolitis. There arecurrently no effective therapies for lung fibrosis due to poorunderstanding of the disease mechanisms.

The (myo)fibroblast has a central role in all types of lung fibrosis³.Ongoing damage to the lungs leads to initiation of dysregulated repairmechanisms with recruitment of fibroblasts and their transformation tomyofibroblasts. Myofibroblasts are the key effector cells in fibrosisproducing excessive amounts of extracellular matrix components likecollagens. Myofibroblasts are currently thought to be the most importanttherapeutic target for treatment of lung fibrosis³. Key growth factorsin the proliferation and transformation of myofibroblasts aretransforming growth factor beta (TGFβ), platelet-derived growth factor(PDGF), and endothelin-1 and many new therapies have focused oninhibiting these factors or their receptors. However, most of theclinical trials based on modulating myofibroblast behaviour have showndisappointing results.

Interferon gamma (IFNγ) is probably the most-studied anti-fibroticmediator in clinical trials of IPF. It mediates myofibroblast growtharrest and apoptosis through a STAT-1 (Signal Transducers and Activatorsof Transcription-1)-dependent pathway, which is important for resolutionof fibrogenic responses⁴. Despite an initial promising start, the largeINSPIRE trial concluded that IFNγ did not prolong survival⁵. IFNγ,however, was administered subcutaneously and since IFNγ receptors arefound on almost all cells in the body, it has severe dose-limiting sideeffects. These include influenza-like illness and fatique⁵. Inhaledadministration can partly circumvent this problem and one trial iscurrently registered to study the effects of aerosolized IFNγ in IPF.

Another way to increase the concentration of IFNγ within myofibroblasts,and thus the efficacy, is to use the concept of drug targeting: drugsare coupled to cell-selective drug carriers that are specifically takenup by target cells, the drugs are released within those cells therebyreducing systemic side effects while attaining high local concentrationsin target cells. Myofibroblasts for instance specifically upregulatePDGF-receptors that can be used for drug targeting purposes⁶.

Although CCl₄ induces liver fibrosis, the lungs also have low expressionof Cytochrome P450 activity and CCL₄ (turned into toxic compounds bythis enzyme) is therefore also slightly activated in lung tissue. Theseleads to activation of profibrotic cells which in turn express the PDGFreceptor, which initiates profibrotic activity. This model has been usedto as a model of idiopathic pulmonary fibrosis (IPF, see ref 8 and 9).While exploring the antifibrotic effects of INF_(Y) in liver tissue, asignificant change in lung tissue was noted by the examiners. The weightof lungs from CCl₄-treated mice was significantly higher than lungs ofnormal mice (0.75±0.05% of body weight in normal versus 2.03±0.15% ofb.w. in untreated CCl₄-treated mice; P<0.01) yet this increase wassignificantly reduced by treatment with PPB-PEG-INF_(Y) (1.53±0.11% ofb.w.; P<0.05 vs untreated CCl₄ mice). Microscopical examination revealedthat the lungs of CCl₄-treated mice were affected by diffuse alveolitis,a condition which can precede fibrosis, and lungs displayed an enhancedcollagen staining. When these mice were treated with IFNγ coupled toPDGF-receptor recognizing peptides, we found a significantly reducedalveolitis in lungs and a reduced collagen deposition. Of note, thesebeneficiary effects of targeted IFNγ were attained after establishingthe lung disease.

We therefore conclude that PDGF-receptor-targeted INF_(Y) is able toaccumulate in any tissue with significant PDGFβ-receptor expression, andis able to exert an antifibrotic effect in other tissues as well asillustrated by its effect in lungs. The INF_(Y) analogs of the inventionmay thus be used for the treatment of idiopathic fibrosis and otherforms of fibrosis or sclerosis in other tissues characterized byenhanced expression of the PDGF-receptor.

Example 11 Targeting of Truncated IFN Gamma to Various Receptors

In this experiment it is shown that an analog of the inventioncomprising the signaling moiety of murine IFNγ (mimiFNγ) and a cellsurface targeting domain can be efficiently targeted not only to thePDGF receptor but also to other cell surface receptors. Exemplarytargeting domains tested include lactose (ligand for theAsialoglycoprotein (ASGP)), mannose (ligand for the mannose receptor (CD206) and the tripeptide RGD (ligand for the receptor avβ3 integrinreceptor). The sequence of 30 mimiFNγ consisted ofFEVNNPQVQRQAFNELIRVVHQLLPESSLRKRKRSR(SEQ ID NO: 36). Various cell typesand different parameters for testing IFNγ activity used in the study.The control samples were exposed to intact murine INNγ. For details seethe Table below.

Parameter measured Target (details IFNγ analog receptor Cells used seetext) Results Lactose-HSA- Asialogly- Human ICAM 1 enhanced PEG-mimIFNγcoprotein Hepatocytes expression expresion (ASGP) (HepG2) vs controlreceptor INFγ Mannose-HSA- mannose Mouse RAW MHC II equal PEG-mimIFNγreceptor macrophages expression expression (CD 206) vs control INFγRGD-PEG- αvβ3 - Endothelial In vitro Enhanced mimIFNγ receptor cellsangiogenesis effectivity assay vs control INFγExperiments with Lactose-HSA-PEG-IFNγ:Synthesis

Lactose-HSA (25 lactose molecules coupled to human serum albumin) wasconjugated to bifunctional PEG molecule (2 KDa). Following dialysis,lactose-HSA-PEG was coupled to SATA-modified truncated IFNγ derived frommouse. The synthesized product (Lac-HSA-PEG-mimIFNγ) was extensivelydialysed against PBS.

Experiment in Human Hepatocytes:

Human Hepatocytes (HepG2) were plated in 12 well plates (1×10⁵cells/well). The cells were grown overnight in 5%/37° C./CO₂ incubator.Subsequently, cells were incubated with medium, mouse IFNγ (1 ug/ml),Human IFNγ (1 ug/ml), mouse IFNγ derived Lac-HSA-PEG-IFNγ (1 ug/ml),Lac-HSA or Lac-HSA-PEG-IFNγ (1 ug/ml) after 2 hrs of blocking withLac-HSA. After 24 hrs of incubation, cells were lysed, RNA was isolatedand reverse transcribed. The cDNA was used for the analysis ofInter-Cellular Adhesion Molecule 1 (ICAM1) expression, which is known tobe induced in response to IFNγ. 18srRNA was used a housekeeping control.

Results:

As expected, neither mouse truncated IFNγ nor Lac-HSA induced ICAM1expression in human hepatocytes, while both human IFNγ and mouse derivedLac-HSA-PEG-IFNγ upregulated ICAM1 expression in human hepatocytes.Furthermore, Lac-HSA-PEG-IFNγ-induced ICAM1 expression was almostcompletely blocked by excess of Lac-HSA.

Conclusions:

The species-specificity of INF_(Y) was confirmed by showing that mouse(truncated) INF_(Y) was ineffective in human hepatocytes. However, thiscytokine was turned into a bioactive compound in human cells aftercoupling to the target moiety lactose, which is known to enterhepatocytes via the Asialoglycoprotein (ASGP) receptor. Specificity forthe ASGP-receptor was shown by blockade of this receptor by Lac-HSA.This demonstrates that the signaling part of INF_(Y) (which is notspecies specific) can be delivered into the cytoplasm of other targetcells than fibroblasts via another target receptor than thePDGF-receptor using a sugar moiety instead of a peptide. Hepatocytetargeting is particularly relevant for the treatment of Hepatitis B andC and ICAM-1 upregulation is a physiological response to enhanceantiviral activity. This experiment supports the use ofcell-specific-truncated INF_(Y) according to the invention as anantiviral compound.

Similarly, mannose was coupled to truncated mouse INF_(Y) according tostandard techniques (L. Beljaars et al J. of Hepatology 29: 579-588,1998) and bioactivity of this mannosylated INF_(Y) was demonstrated inmouse macrophages with MHC class II expression as read-out parameters(rtPCR methods).

The endothelial binding peptide RGD—or its control peptide RAG, thatdoes not bind to these cells, were also coupled to truncated mouseINF_(Y) according to standard methods. The resulting analogs wereevaluated for their biological activity in cultures of endothelial cells(H5V) by measuring tube formation which is a parameter reflectingangiogenesis. Tube formation by H5V cells was inhibited most potently byRGD-mimINF_(Y) in this assay (data not shown).

Conclusions:

Modification of truncated mouse INF_(Y) by conjugation to a targetingmoiety (e.g. an oligosaccharide or peptide) is feasible withoutdisturbing the bioactivity of the signaling part of this cytokine.Delivery of an INF_(Y) analog which is defective in binding to itsnatural receptor but which instead can bind to a distinct cell surfacereceptor mediating cellular uptake of the analog allows for moreeffective therapeutic applications with less side-effects.

References

-   1. du Bois R M. Strategies for treating idiopathic pulmonary    fibrosis. Nat Rev Drug Discov; 9(2):129-40.-   2. Wilson M S, Wynn T A. Pulmonary fibrosis: pathogenesis, etiology    and regulation. Mucosal Immunol 2009; 2(2):103-21.-   3. Scotton C J, Chambers R C. Molecular targets in pulmonary    fibrosis: the myofibroblast in focus. Chest 2007; 132(4):1311-21.-   4. Bonner J C. Mesenchymal cell survival in airway and interstitial    pulmonary fibrosis. Fibrogenesis Tissue Repair; 3:15.-   5. King T E, Jr., Albera C, Bradford W Z, et al. Effect of    interferon gamma-1b on survival in patients with idiopathic    pulmonary fibrosis (INSPIRE): a multicentre, randomised,    placebo-controlled trial. Lancet 2009; 374(9685):222-8.-   6. Beljaars L, Weert B, Geerts A, Meijer D K, Poelstra K. The    preferential homing of a platelet derived growth factor    receptor-recognizing macromolecule to fibroblast-like cells in    fibrotic tissue. Biochemical pharmacology 2003; 66(7):1307-17.-   7. Homma S, Nagaoka I, Abe H, et al. Localization of    platelet-derived growth factor and insulin-like growth factor I in    the fibrotic lung. Am J Respir Crit Care Med 1995; 152(6 Pt    1):2084-9.-   8. Paakko P, Anttila S, Sormunen R, et al. Biochemical and    morphological characterization of carbon tetrachloride-induced lung    fibrosis in rats. Arch Toxicol 1996; 70(9):540-52.-   9. Mizuguchi S, Takemura S, Minamiyama Y, et al. 5-allyl cysteine    attenuated CCl4-induced oxidative stress and pulmonary fibrosis in    rats. Biofactors 2006; 26(1):81-92.

Example 11 Binding Study of BiPPB to Cultured or Freshly IsolatedPrimary Cells

This example shows that Bi-PPB either produced chemically or throughrecombinant techniques specifically binds to the PDGFβ-receptor.Receptor specificity is demonstrated by blocking the binding withspecific antiPDGF-β-receptor antibodies. BiPPB is species non-specificas it binds to Myo-fibroblast-like cells of rat, mouse and human.Receptor interaction requires at least two cyclic peptides, as themonocyclic form does not bind to the target receptor.

Sequence of BiPPB:

C(1)S R N L I D C(1)G G G D G G C(2)S R N L I D C(2)(SEQ ID NO: 59):Cys(1)-Cys(1) and Cys(2)-Cys(2) disulfide bridge cyclisationsMethods:

The binding of BiPPB was performed in primary freshly isolated rathepatic stellate cells. Cells were seeded in the 8-well glass plates(Lab-Tek, Nunc, Naperville, Ill.) at 30,000 cells/well in the culturemedium. After overnight incubation at 37° C./5% CO2, cells were washedwith PBS and subsequently incubated with FITC-labeled PPB (monocyclic)or BiPPB (bicyclic: 10 μg/ml) at room temperature. To block the binding,anti-PDGF-βR IgG was added to the cells 1 h before FITC coupled PPB orBiPPB. After 2 h, cells were washed 3 to 4 times with cold PBS and fixedwith 4% paraformaldehyde. The nuclei were counterstained with DAPI andmounted in citifluor (anti-fade reagent) and visualized underfluorescent microscope.

Similar binding experiments were performed in primary freshly isolatedhuman myofibroblasts, mouse 3T3 fibroblasts and human hepatic stellatecells (LX2). For human Hepatic stellate cells, the sequence of BiPPB wasas follows:

C(1)S R N L I D C(1) KGSGSGG C(2)S R N L I D C(2)(SEQ ID NO: 60):Cys(1)-Cys(1) and Cys(2)-Cys(2) disulfide bridge cyclisationsResults:

The FITC-coupled monocyclic PPB did not show any binding as thePDGF-receptor requires dimeric interaction. FITC-coupled BiPPB showedsignificant binding to the cell type tested, which was almost completelyblocked by PDGF receptor antibody, showing the receptor specificity ofthe binding to these cells (data not shown).

The invention claimed is:
 1. An analog of interferon gamma (IFNγ) theanalog comprising the amino acid sequence set forth in SEQ ID NO: 56 orthe amino acid sequence set forth in SEQ ID NO:
 58. 2. The analogaccording to claim 1, further comprising at least one non-antigenicpolymer.
 3. A pharmaceutical composition comprising an analog accordingto claim 1 and a pharmaceutically acceptable carrier, adjuvant and/orexcipient.